The incidence of the Inflammatory Bowel Diseases (IBD), Crohn's Disease (CD) and Ulcerative Colitis (UC) has increased dramatically over the past four decades with approximately five million individuals affected in North America and Europe. While therapeutic options have increased over the past decade, the ability to classify subtypes of IBDs, predict disease progression and behavior, and target newer biologic therapies to specific subgroups of patients has lagged behind. This has led to an empiric step-up approach to therapy, in which increasingly more potent agents are offered until an effective regimen is identified.
The inflammatory bowel diseases are believed to be caused by a complex interaction between genetic susceptibility and environmental triggers leading to chronic relapsing intestinal inflammation (Bouma, G. 2003). CD is characterized by discontinuous transmural inflammation which can involve any part of the gastrointestinal (GI) tract, although the terminal ileum and proximal colon are most commonly involved (Hanauer S. B. 2006). Conversely, UC is characterized by continuous superficial inflammation limited to the colon, beginning in the rectum and extending proximally (Hanauer S. B. 2006).
IBDs can be difficult to diagnose, a diagnosis being obtained only after extensive, costly, and often invasive procedures have been performed. An initial diagnosis, made on the basis of medical history and physical examination, is generally confirmed via imaging of the intestines and laboratory culture tests to rule out bacterial, viral and parasitic infections. Colonoscopy may also be used to image the intestines and colon, such that the entire intestines can be evaluated to distinguish between IBDs on the basis of the location of ulcerations (Crohn's disease affects some areas of the intestines and skips over others, while ulcerative colitis is more indiscriminate). Endoscopy may also be used to biopsy the intestinal tissue, which can be used to identify the deep inflammation of the bowel that is characteristic of Crohn's disease. Further, X-rays (after oral or rectal ingestion of Barium), computed tomography (CT) scan, and magnetic resonance imaging (MRI) may be helpful in locating fistulas and assisting in the diagnosis. A stool analysis (including a test for blood in the stool) is often performed, depending on symptoms, to look for blood and signs of bacterial infection. Blood and urine tests may be done to check for anemia, high white cell counts, or malnutrition—all signs of IBDs. Finally, the conditions may go undiagnosed for years as symptoms usually develop gradually and less than all of the intestines may be involved. As such, while there is currently no reliable biochemical test available for IBD or to distinguish subtypes of the disease, such a test would be highly valuable in curtailing cost and the physical discomfort endured by individuals in obtaining an IBD diagnosis.
With current diagnostic approaches, approximately 60% of IBD patients are classified as CD, 30% as UC, and 10% as indeterminate colitis (IC) (Kugathasan S. 2003). Patients with IC have intestinal inflammation limited to the colon, without specific endoscopic or histological features diagnostic of either CD or UC. A diagnosis of IC limits the ability to predict clinical disease behavior and outcome following surgery, and prevents patients with refractory disease from entering into clinical trials of new agents.
IBD causes substantial morbidity including frequent hospitalizations and surgeries, and longstanding disease is complicated by cancer (Hanauer S. B. 2006). It is likely that there are several immunogenetic sub-types of IBD, with CD and UC representing the broadest clinical classifications (Dubinsky M. C. 2006). Current evidence suggests that CD results when mucosal tolerance to the enteric flora is lost (Bouma G. 2003, Lodes M. J. 2004). CARD15/NOD2, an intra-cellular sensor for the bacterial product muramyl dipeptide, is expressed in intestinal epithelial cells (IEC) and macrophages and participates in anti-microbial defenses (Houma G. 2003, Kobayashi K. S. 2005). Loss of function mutations in NOD2 increases susceptibility to CD primarily involving the ileum (Tomer G 2003).
A variety of defects in neutrophil function have also been described in CD (Korzenik J. R. 2000). These primary defects in mucosal innate immunity may then lead to activation of intestinal antigen presenting cells (APG) and expansion of Teff reactive to the host flora (Bouma G. 2003, Lodes M J 2004). The immunogenetic basis for differences in ileal versus colonic involvement in CD is not known, although mounting evidence suggests that defects in innate immunity and loss of tolerance to bacterial antigens are predominately associated with small bowel disease. Mutations in CARD15 are associated with ileal disease and growth failure in children, while seroreactivity to microorganism components (ASCA, OmpC, CBir1, and I2 antibodies) is associated with small bowel, stricturing disease (Tomer G 2003), (Mow W. S. 2004). This does not appear to be the case for UC, in which defects in epithelial repair and xenobiotic detoxification are implicated (Dignass A. U. 2004). The introduction of biologic therapies targeting specific components of the mucosal immune response has improved outcomes for patients with IBD. However, sustained remissions have not been observed in more than fifty percent of individuals with any new biologic agent.
Current Therapies for Inflammatory Bowel Diseases
Current options for induction of remission in IBD include mesalamine, corticosteroids, methotrexate, and infliximab. Options for maintenance of remission include mesalamine, the immunomodulators 6-mercaptopurine/azathioprine (6-MP/AZA), methotrexate, and infliximab. The most common first line regimen includes induction of remission with prednisone, and maintenance of remission with 6-MP/AZA. In the absence of a reliable test to predict response to therapy, patients are empirically offered agents for induction and maintenance of remission largely based upon disease severity and location. As the effectiveness of any one agent is typically on the order of 50% to 80%, this leads to a substantial number of patients receiving a series of ineffective agents, with attendant side effects, before an effective regimen is identified.
Despite the recent advent of the biologic therapies, corticosteroids remain the predominant first line choice for induction of remission in moderate to severe IBD. Typically, 50% of patients will go into remission and then be able to wean corticosteroids within three months. However, 20% will not respond, and 30% will not be able to discontinue steroids; these groups are termed steroid refractory (SR) and steroid dependent (SD), respectively.
Inflammatory Bowel Disease Markers for Diagnosis and Progression of Disease
In recent years, serological markers which largely comprise antibodies reactive to the host flora have been extensively studied in an attempt to improve IBD classification and prediction of disease progression. However, these markers have not proven effective in definitively characterizing IBD patients as CD or UC, and have not been able to predict with sufficient accuracy which patients are most likely to require surgery for the condition.
The first serological markers to be described in IBD were the anti-saccharomyces cervisiae antibody (ASCA) and anti-neutrophil cytoplasmic antibody with perinuclear staining (pANCA) (Austin G. L. 2006). Subsequently, additional markers including antibodies to the outer membrane porin protein C (OmpC) of E. Coli, to Pseudomonas fluorescens (I2) and to flagellin (CBir1) have been described (Dubinsky M. C. 2006). Whether any of these antibodies are pathogenic is not known, although recent evidence points to bacterial flagellin as a critical antigen in CD (Lodes M. J. 2004). pANCA has been associated with UC, while the other markers have been associated with CD. A recent series of well-characterized adult CD patients demonstrated a frequency of 37% for OmpC, 52% for I2, 39% for ASCA, and 14% for pANCA (Arnott I. D. 2004). The frequency of pANCA in UC is typically 60% to 70% (Austin G. L. 2006). Multiple studies have been performed to determine whether levels of these antibodies can be used to screen for IBD in patients with suggestive symptoms, and to discriminate CD from UC, thereby reducing the number of cases of IC. However, the sensitivity of pANCA for UC in recent adult and pediatric series has ranged from 63% to 70%, while the sensitivity of ASCA for CD ranged from 44% to 72% (Linskens R. K. 2002), (Zholudev A. 2004), (Gupta S. K. 2004). Thus, these results demonstrate that these markers are not sufficiently sensitive to be used as effective screening tools. In terms of specificity, these assays perform substantially better, and range from 86% to 95% for pANCA for UC and from 82% to 95% for ASCA for CD (Linskens R. K. 2002, Zholudev A. 2004, Gupta S. K. 2004). From 10% to 18% of CD patients will be positive for pANCA, these typically have Crohn's colitis (Zholudev A. 2004). In cohorts of patients with a high prevalence of IBD (42% to 68%), this has led to a reported positive predictive value (PPV) of 90% to 96% for both CD and UC, and a negative predictive value (NPV) of 50% to 80% (Austin G. L. 2006, Gupta S. K. 2004). Therefore, as recently reviewed by Austin et al, these tests perform best when used in a patient population with a high pre-test probability of having IBD (Austin G. L. 2006). As such, there is a need for improved markers having improved sensitivity and selectivity that can be used as effective screening tools to diagnose and differentiate inflammatory bowel diseases.
IBD patients further have variable risk of needing surgery to treat the disease. Current standard clinical approaches are not able to further reduce the number of diagnoses of IC, or to predict which patients will be most likely to progress to surgery. From about 30% to 50% of IBD patients will fail medical therapy and progress to surgery within 3 to 10 years of diagnosis (Austin G. L. 2006). Most CD patients initially present with inflammatory disease (80%), and over the next 10 years may progress to stricturing and/or penetrating behavior, categorized as B2/B3-type disease (Hanauer S. B. 2006). Patients with stricturing/penetrating disease then frequently require surgery.
Several groups have performed studies to determine whether IBD serology can predict disease progression and the need for surgery (Mow W. S. 2004). Consistently, high titer ASCA+CD patients have been more likely to have fibrostenosing small bowel disease and require ileocecal resection (36% vs 13% in one recent pediatric series) (Arnott I. D. 2004). Conversely, pANCA+CD patients are more likely to have colonic disease and not require resection. Recent reports demonstrated that the combined magnitude of reactivity to microbial components (ASCA, OmpC, and I2) predicted a phenotype of more severe small bowl CD with progression to surgery in adult patients (Arnott T. D. 2004). However, the magnitude of the serological response did not predict the need for 6-MP or infliximab. Importantly, the association was also independent of the CARD15 genotype, which has also been associated with small bowel CD and progression to surgery (Arnott I. D. 2004, Walker L. K. 2004).
Recent reports in pediatrics have demonstrated very similar predictive characteristics High titer ASCA+CD patients were more likely to require surgery sooner than ASCA-patients (Amre D. K. 2006). In a study measuring ASCA, OmpC, I2, and CBir1 reactivity, the presence and magnitude of response of these was associated with a higher rate of progression to a stricturing or penetrating complication over the first 18 months following diagnosis (Dubinsky M. C. 2006). Potentially this sub-group of CD patients would benefit from earlier institution of targeted biologic therapies more likely to prevent disease progression. In this regard, Mow et al have recently reported a trend towards improved responses to antibiotics in CD patients sero-reactive to OmpC and/or I2, and decreased responses to a steroid preparation, budesonide (Mow W. S. 2004).
A limitation of this approach is the substantial number of patients who experience disease progression and require surgery who are sero-negative for these markers. For example, in the Arnott report, 96 CD patients were negative for ASCA or OmpC/I12, and fifty percent of those experienced disease progression and required surgery (Arnott I. D. 2004). This compared to 40 CD patients who were sero-positive, with rates of disease progression and surgery of 90% and 75%, respectively (Arnott I. D. 2004). Therefore, additional approaches are needed to complement current serological assays in defining clinically important sub-types of IBD with respect to disease behavior and progression.
Current biomarkers have failed to be effectively used to provide diagnostic tests for classifying, characterizing or predicting the outcome of inflammatory bowel diseases. In particular, current biomarkers have not been efficacious in distinguishing among the various subtypes of inflammatory bowel diseases. Accordingly, there is a need for biomarkers that can form the basis for diagnostic tools that can effectively classify, characterize and predict the severity of inflammatory bowel disease.
There is further the need for biomarkers that can be used to predict disease progression, including the likelihood that an individual will require surgery despite treatment with first line therapies would be invaluable. Such markers would allow the clinician to identify the subset of patients at an early stage of disease progression who would benefit from earlier introduction of second or third line therapies, or therapies particularly effective in treatment of a particular subset of IBD, such as anti-TNFα (for example, Remicade or Humira) and/or GM-CSF (Leukine) or the like. The instant invention satisfies these needs and provides related advantages as well.